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Abstract

Distal diabetic peripheral polyneuropathy (DPN) is a prevalent complication resulting from chronic hyperglycemia in diabetic patients. It is associated with incapacitating pain, foot ulceration, and lower-limb amputations and brings about physical and psychological burdens to a patient's quality of life and a large economic burden to the health care system. Despite the prevalence and severity of DPN, the development of therapies that have focused on "diabetes specific" targets has met with limited translational success. This is due, at least in part, to the fact that disease progression among individuals does not occur with temporal and/or biochemical uniformity. Thus, our innovative approach has explored the premise that it is not necessary to target a specific pathogenic mechanism to reverse DPN and that pharmacologic induction of cytoprotective molecular chaperones affords a novel mechanism to improve myelinated and unmyelinated fiber function in DPN. To this end, we established that KU-32, a novel, non-toxic small molecule inhibitor of the molecular chaperone heat shock protein 90 (Hsp90) was able to protect sensory neurons from glucotoxicity and decrease the symptoms of DPN in diabetic mice. However, despite KU-32's efficacy in improving the sensory deficits of DPN in diabetic mice, specific mechanisms of neuroprotection remain unidentified. Thus, this dissertation utilized variations of stable isotope labeling with amino acids in cell culture (SILAC) as a novel, unbiased and systematic approach to quantitatively study the effect of hyperglycemia and KU-32 on the mitochondrial proteomes from dorsal root ganglia (DRG) neurons and Schwann cells (SCs) as in vitro models of DPN. This dissertation provides the first quantitative characterization of the temporal effect of hyperglycemia on the SC proteome using SILAC. Specifically, hyperglycemia increased the expression of numerous mitochondrial proteins in SCs that regulate oxidative phosphorylation and anti-oxidant responses. Consistent with these observations, hyperglycemia did not induce superoxide production in SCs and this correlated with an increase in MnSOD and the extent of proton leak, which may function in reducing oxidative stress. Although hyperglycemia increased mitochondrial respiration, the increase in proton leak suggests that respiration is less efficient at producing ATP. However, this deficiency may be offset by the ability of hyperglycemia to increase glycolysis in SCs. In contrast, hyperglycemia decreased mitochondrial respiratory capacity in DRG sensory neurons and promoted a robust induction in superoxide production. Treatment of KU-32 antagonized the effect of hyperglycemia by decreasing mitochondrial superoxide levels and improving organellar bioenergetics in DRG neurons. These functional improvements correlated with the translational induction of MnSOD and mitochondrial chaperones by KU-32. Overall, studies in this dissertation provide evidence that sensory neurons and SCs have rather distinct energetic responses to hyperglycemia and that SCs can more effectively decrease glucose-induced oxidative stress. These studies also provide proof-of-principle that pharmacological induction of molecular chaperones may ameliorate DPN by helping sensory neurons to decrease oxidative stress and improve mitochondrial bioenergetics in response to glucotoxicity.